Periodontitis is a chronic inflammatory illness that damages the bone surrounding periodontal tissue and is brought on by the buildup of bacterial plaque along the gingival border (Haririan et al., 2014; Loos and Van Dyke, 2020). It is an infectious illness that may aggravate or lessen various risk factors (Loos and Van Dyke, 2020). By producing large quantities of virulence factors that modify host immune responses, some bacteria, such as Porphyromonas gingivalis, a common cause of periodontal inflammation, are responsible for the production of periodontitis in susceptible individuals (Armingohar et al., 2014; Loos and Van Dyke, 2020). The osteoclast differentiation factor receptor activator of nuclear factor-B ligand (RANKL) is induced by P. gingivalis (Belibasakis et al., 2011). When monocytes and macrophages secrete inflammatory cytokines in response to RANKL stimulation, several proteolytic enzymes are produced, promoting bone resorption (Ono et al., 2020). Animal models infected with this acute periodontal disease have been used to study bone remodeling mechanisms (Yang et al., 2019).

CBD is a chemical compound derived from the cannabis plant. It is one of the many phytocannabinoids found in the plant, along with tetrahydrocannabinol (THC), a compound responsible for the psychoactive effect of cannabis (Campos et al., 2012). CBD has gained increasing attention recently due to its potential therapeutic properties and minimal side effects. Research suggests that CBD may have anti-inflammatory (Iffland and Grotenhermen, 2017), analgesic, anxiolytic, and neuroprotective effects (Lim et al., 2017). These properties make it a potential treatment option for anxiety, depression, chronic pain, epilepsy, and neurodegenerative diseases.

Taurine is a sulfur-containing amino acid found in various tissues throughout the body (Schuller-Levis and Park, 2004). Taurine is well-known for possessing antioxidant properties (Lim et al., 1998). Taurine is also involved in tissue repair in periodontal disease (Özmeriç et al., 2000). Taurolidine, a taurine derivative, has been suggested as a potent antimicrobial agent in non-surgical and surgical periodontal treatments (Eick et al., 2012).

This study was designed to test the hypothesis that CBD plus taurine may have synergic and beneficial effects in inhibiting bone resorption and treating periodontitis in a rat model.

Chemical and reagents

The CBD were obtained from Jeonbuk National University LED Agricultural Life Science Convergence Technology Research Center (Jeonju, Korea) in solid state. It was dissolved in dimethyl sulfoxide (DMSO) and diluted to the required working concentration. Taurine was purchased from Sigma Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin, and streptomycin were purchased from Gibco (Grand Island, NY, USA). The cell counting kit-8 (CCK-8) was obtained from Dojindo Molecular Technologies (Rockville, MD, USA). The Leukocyte Acid Phosphatase (TRAP) kit was purchased from Sigma Aldrich. Calcium phosphate (CaP) coated plates were purchased from Cosmo Bio (Tokyo, Japan). Recombinant soluble mouse RANKL was purchased for R&D (R&D systems, MN, USA). Primary and secondary antibodies were obtained from Cell signaling technology (Danvers, MA, USA) and Santa Cruz biotechnology (Santa Cruz, CA, USA).

Animals

The ethics committee for animal handling at Jeonbuk National University (Jeonju, Korea) approved the study. All rat experiments were performed in accordance with the local institutional Animal Care and Use Committee (IACUC) guidelines. Six-week-old male Sprague-Dawley rats were obtained from Nara Biotech Pyeongtaek Plant (Pyeongtaek, Korea). All rats were housed on a 12-h light and 12-h dark schedule and were single-housed with water and food in standard plastic cages at 24°C-26°C.

Periodontal disease rat model and timeline design

Our experimental animal periodontitis model was established in accordance with previously described protocols (Yang et al., 2021). The rats were divided into the following 5 groups: healthy (G1), vehicle (G2), 2 mg/kg CBD+100 mg/kg taurine (G3), and 20 mg/kg CBD+100 mg/kg taurine (G4). Ligatures were immersed overnight in a P. gingivalis culture (10⁹/mL) at controlled room temperature (25 ± 2°C). P. gingivalis ligatures were placed in the first and second teeth of each rat’s left maxilla for 7 days. Anesthesia was administered intramuscularly as a 20 mg/kg Zoletil 50 injection. On day 7, rats from all groups were examined for periodontal pocket depths. The periodontal pocket depths were monitored daily between days 14 and 17 in the treatment groups (G3, G4, and G5) as they received treatment daily for 14 days. The rats in G1 and G3–4 were sacrificed on day 20, and blood serum, periodontal pocket tissue, and alveolar bone samples were collected.

Culture of macrophages

Murine macrophage cells were cultured by previously described protocols (Li et al., 2021). DMEM supplemented with FBS (10%), penicillin (100 U/mL), and streptomycin (100 U/mL) antibiotics at 37°C and 5% CO₂, RAW264.7 (ATCC, Manassas, VA, USA) cells were cultured as murine macrophages. BMMs were isolated from the tibiae and femurs of mice that were 6 weeks old (Nara Biotech Pyeongtaek Plant). Adhering cells were harvested and grown for 3 days in Minimum Essential Medium Eagle with 10% FBS, macrophage colony-stimulating factor (30 ng/mL), and RANKL (50 ng/mL).

Cell viability assay

Cell viability was measured following previously described protocols (Li et al., 2016). To determine the impacts of CBD and taurine on the viability of RAW 264.7 cells, a cytotoxicity assay was carried out using the Cell Counting Kit-8 following the manufacturer’s instructions. In 96-well plates, RAW264.7 cells (5×10³ cells/well) were implanted for 16 h before being exposed to various dosages of CBD and taurine for 1, 3, or 6 days. After exposing the cells to the CCK-8 solution for two hours, the optical density at 540 nm was measured using a microplate reader (Power Wave HT, Biotek, Santa Clara, CA, USA). The cell viability was calculated as a percentage of the control, and the findings are shown as mean ± SD of triplicate wells.

TRAP staining

The method of tartrate-resistant acid phosphatase (TRAP) staining was prepared as described previously (Park et al., 2022). In each well of a 96-well plate, 3×10³ RAW264.7 cells were plated and incubated with RANKL (50 ng/mL) and CBD, taurine, or a combination of the two. The mixture was changed every 2 days. After 5 days, the cells were washed with phosphate buffer saline (PBS) and fixed with 3.7% formalin for 10 min at room temperature. The fixed cells were washed with PBS, incubated with 0.1% (V/V) Triton X-100 for 1 min, and then rewashed with PBS. The samples were stained by TRAP according to the manufacturer’s instructions. TRAP-positive multinucleated cells containing ≥ nuclei were counted under light microscopy.

Pit formation assay

The bone resorption pit assay was carried out using a Bone Resorption Test Kit (CSF-BRA-48 KIT, Cosmo Bio) in plates coated with calcium phosphate. For that, 5×10³ BMMs were plated on 48-well CaP plates and treated with 30 ng/mL macrophage colony stimulating factor (M-CSF) 50 ng/mL RANKL, and a dose of 10 μM CBD, 0.5 mM taurine, and a combination of 10 μM CBD and 0.5 mM taurine for 6 days. The culture media were changed every 2 days. The cells were stained using a TRAP staining kit, and the pit area was evaluated was calculated as defined earlier (Li et al., 2016).

Enzyme-linked immunosorbent assay

Rats were used to collect serum samples for tumor necrosis factor-alpha (TNF-α) and interleukin -1 beta (IL-1β) measurement. According to the manufacturer’s recommendations, the enzyme-linked immunosorbent assay (ELISA) kits were used to quantify the blood levels of TNF-α and IL-1β.

Western blots

The method of protein extraction and western blot analysis was performed as described previously (Park et al., 2022). After washing with PBS, the RAW264.7 cells were lysed in ice-cold buffer containing sodium deoxycholate (0.25%), EDTA (1 mM), NaCl (150 mM), Tris-HCl (50 mM), aprotinin (5 µg/mL), Na₃VO₄ (1 mM), pepstatin (1 µg/mL), NaF (20 mM), NP-40 (1%), and phenylmethylsulfonyl fluoride (1 mM). After 30 min, the lysates were collected by centrifugation (16,100 RCF at 4°C for 10 min). Using Tris-buffered saline with Tween-20 (0.25%) at 16°C (TBST), the transferred polyvinylidene difluoride membrane was blocked in nonfat skim milk (5%), followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10%) to separate the proteins. The membrane was incubated with anti-iNOS and anti-COX-2 (Santa Cruz Biotechnology, Dallas, TX, USA) at a dilution of 1:2,000 to 1:3,000 in nonfat skim milk in TBST for 18 h at 4°C. Then, the membrane was incubated for 2 h with a secondary antibody diluted between 1:2,500 and 1:3,000 in dry milk in TBST. Western blotting agents with increased chemiluminescence were used to create the blots (Sigma Aldrich).

Measurement of alveolar bone loss

The buccal and palatal surfaces of the upper second molars of all mice were measured for the distance between the cementoenamel junction (CEJ) and the alveolar bone crest (ABC) to assess alveolar bone loss.

Histomorphometric analysis

Maxilla samples were extracted and fixed in 10% formalin-containing 0.1 M phosphate buffer at a pH of 7.4 overnight, and 10% EDTA (changed every 3 to 5 days) was decalcified in the samples for 4 weeks. The dehydrated and decalcified specimens were paraffin-embedded, cut into 5 µm sections, stained with hematoxylin and eosin, and examined under light microscopy. The distance between the CEJ and ABC was measured in the proximal region of the upper molars.

Statistical analysis

Three experiments were conducted, and one-way ANOVA with Tukey’s multiple comparisons test was used to assess the results. The data are presented as mean ± SD and were analyzed using the GraphPad Prism program (GraphPad Prism Inc., La Jolla, CA, USA). We considered values of p<0.05 to be statistically significant.

Cytotoxicity of CBD and taurine in RAW264.7 cells

To examine CBD and taurine effects on cell viability, the cells were pretreated under various doses of CBD, taurine, or their combination for 72 h and evaluated by CCK-8 assay. We observed that CBD up to 12 µM and taurine up to 0.6 mM did not result in significant cell death (data not shown).

Suppression of iNOS and COX-2 protein expression

To explore the anti-inflammatory effect of CBD and taurine, RAW264.7 cells were treated with or without CBD, taurine, or the combination. Cells were treated with CBD, taurine, and a combination of CBD and taurine for 30 min, after then 2 μg/mL LPS was treated with the cells for 12 h and then protein was isolated from each group. Fig. 1 shows that stimulation with LPS significantly increased the iNOS and COX-2 protein expression in RAW264.7. However, combined treatment of CBD and taurine downregulated LPS-induced protein levels compared with the LPS-only, CBD-only, and taurine-only groups. This result suggests that combining the treatment of CBD and taurine may exert anti-inflammatory responses by suppressing LPS-induced expression of inflammatory markers.

Figure 1. Effect of CBD, taurine, and combining CBD and taurine on the production of iNOS and COX-2 on LPS-stimulated expression in RAW264.7 cells. The protein levels of LPS-induced iNOS and COX-2 were expressed by western blot. (A-C) RAW264.7 cells were pretreated with the indicated concentration of CBD, taurine, and both for 30 min, then stimulated with 2 µg/mL of LPS for 12 h. Resulted are presented as means ± SD (n=3). **p<0.01, ***p<0.001 indicates significant differences from CBD+taurine treated groups with CBD and taurine alone.

Effects of CBD and taurine on LPS-induced pro-inflammatory cytokines

Generally, TNF-α and IL-1β are released by LPS-stimulated macrophage cells and are associated with inflammatory responses. ELISA was performed to measure the secreted levels of TNF-α and IL-1β in the culture medium of RAW264.7 cells to perceive the effects of CBD and taurine on inflammation. Both cytokine levels were increased by LPS treatment, but the combination of CBD with taurine significantly reduced this induction in vitro (Fig. 2). This result indicates that combining CBD with taurine can alter the LPS-induced pro-inflammatory activity of cytokines TNF-α and IL-1β.

Figure 2. Effect of CBD, taurine, and combining CBD and taurine on proinflammatory cytokines in LPS-stimulated RAW264.7 cells. The pro-inflammatory levels of LPS-induced TNF-α and IL-1β were expressed by ELISA assay. (A, B) RAW264.7 cells were pretreated with the indicated concentration of CBD, taurine, and both for 30 min, then stimulated with 2 µg/mL of LPS for 12 h. Resulted are presented as means ± SD (n=3). ***p<0.001 indicates significant differences from CBD+taurine treated groups with CBD and taurine alone.

Effects of CBD and taurine on RANKL-induced osteoclast differentiation in RAW264.7 cells

The effects of CBD and taurine in osteoclastogenesis were examined. RAW264.7 cells were incubated with various doses of CBD, taurine, or their combination for 5 days, changing the media every 2 days. Fig. 3A and 3C shows that the multinucleated cells were high in RANKL-treated cells. The combination of CBD and taurine significantly reduced TRAP+ cells in comparison to the untreated and CBD and taurine-only groups. CBD and taurine inhibit the differentiation of RAW264.7 cells into osteoclasts.

Figure 3. Inhibitory effect on osteoclast differentiation and bone resorption. (A) RAW264.7 cells were cultured in DMEM high glucose for 5 days in the presence of RANKL (50 ng/mL) with CBD, taurine, and both. (B) RAW264.7 cells were seeded in CaP-coated 48-well plates and treated with 50 ng/mL RANKL in the presence of CBD, taurine, and both. (C) TRAP-positive multinucleated cells (TRAP+ MNCs) were counted after fixation and staining for TRAP. (D) Pit area was measured. The results are presented as means ± SD (n=3). ***p<0.001 indicates significant differences from CBD+taurine treated groups with CBD and taurine alone.

The combination of CBD with taurine impairs bone resorption

Since CBD and taurine repressed osteoclastogenesis, we examined whether the combination could decrease osteoclast bone resorption activity in calcium phosphate-coated plates. Therefore, we seeded RAW264.7 cells in calcium phosphate-coated plates. As shown in Fig. 3B and 3D, mature osteoclasts in the RANKL-treated group broadly resorbed calcium phosphate in these coated plates. Combining CBD and taurine significantly decreases the resorption activity compared to untreated or single-treatment groups.

Reductions of periodontal bone loss and pocket depth by CBD and taurine

An animal model with P. gingivalis-induced periodontitis was utilized to examine the therapeutic effects of CBD and taurine on alveolar bone loss and periodontal inflammation. Each rat was sacrificed, and the maxilla was obtained. The collected blood sample was separated into serum and analyzed for serum proinflammatory cytokines TNF-α (Fig. 4A) and IL-1β (Fig. 4B). Proinflammatory cytokines were considerably reduced in the H-CBD (20 mg/kg)+taurine (100 mg/kg)-treated group compared to the groups treated with vehicle, and L-CBD (2 mg/kg)+taurine (100 mg/kg). The distances between the CEJ and ABC were then calculated (Fig. 5). According to the histomorphometric analysis, alveolar bone loss was considerably reduced in the H-CBD (20 mg/kg)+taurine (100 mg/kg)-treated group compared to the groups treated with vehicle, and L-CBD (2 mg/kg)+taurine (100 mg/kg). Notably, the H-CBD and taurine combination exhibited a more remarkable effect. As anticipated, the P. gingivalis–infected group showed greater bone loss and periodontal inflammation compared to the healthy control and drug-treated groups. Clinical pocket depth was also assessed between days 14 and 17 to assess the impacts of CBD and taurine on periodontal inflammation. The combination group exhibited significantly narrower pockets compared to the vehicle group (Fig. 5A).

Figure 4. Alveolar bone loss and periodontal pocket depth. Representative images of the distance from cementoenamel junction (CEJ) to alveolar bone crest (ABC) in each group. (A) Black dotted line and the blue dotted lines represent the CEJ and ABC, Red line is the distance from CEJ to ABC, and the round red dotted line represents the bone loss area (Vehicle group) (upper panels, hematoxylin and eosin (H&E) stain, scale bar=200 μm). (B) Measurements of the distance from CEJ to ABC in each group (Sham group, healthy; Vehicle group, induce-ligatured group and only PBS oral administration; L-CBD+Taurine group, 2 mg/kg (Low dose CBD) with 100 mg/kg (taurine) oral administration; H-CBD+taurine group, 20mg/kg (High dose CBD) with 100 mg/kg (taurine) oral administration). (C) Periodontal pocket depth was measured daily from day 7 and 14 to day 20. Results are presented as means ± SD (n=5). P value of less than 0.05 was considered statistically significant. *p<0.05 indicates significant differences from H-CBD+taurine treated groups with vehicle. ^#p<0.05 as compared with the Sham group.

Figure 5. Evaluation of proinflammatory cytokines in rats treated with L-CBD (2 mg/kg) and H-CBD (20 mg/kg) with taurine (100 mg/kg). Serum proinflammatory cytokines TNF-α (A) and IL-1β (B) were measured by ELISA assays. The results are presented as means ± SD (n=3). **p<0.01, ***p<0.001 indicates significant differences from H-CBD+taurine treated groups with L-CBD+taurine.

The most commonly used clinical treatment strategy in the treatment of periodontal disease is the physical removal of biofilm and calculus through scaling (Herrera et al., 2002). Chlorhexidine is an antibiotic used in conjunction with surgical treatment of periodontal disease, and the systemic antibiotics amoxicillin or metronidazole and doxycycline are most commonly used. However, as antimicrobial resistance in human pathogens increases, antibiotic resistance in periodontal disease patients is increasing (Rams et al., 2014). Recently developed periodontitis treatment is used to treat periodontal disease through modulation of immune response. Recently developed periodontitis treatment is used to treat periodontal disease through modulation of immune response. Resveratrol, a natural polyphenol, exhibits immunomodulatory effects against F. nucleatum through inhibition of NF-κB activation in macrophages and upregulation of antioxidant pathways (Lagha et al., 2019; Lim et al., 2020). Metformin reduces inflammation through the regulation of IL-1β (Wang et al., 2020). Catechin is another polyphenol used as an immunomodulator, and in mouse models, catechin is effective against gingivitis by reducing lL-1β in macrophages (Lee et al., 2020). Gliclazide has antioxidant properties and reduces the infiltration of neutrophils and macrophages in a rat model of periodontal disease, and can also reduce inflammation by reducing TNF-α levels (Araujo et al., 2019).

Cathepsin K (Ctsk) can regulate periodontal health by reducing inflammation and osteoclast activity by lowering the levels of TNF-α, INF-γ, IL-1α, IL-1β, and IL-12 (Chen et al., 2016). Strontium ranelate inhibited periodontitis in an estrogen-deficient rat ligature model by increasing the number of osteoblasts, reducing the number of osteoclasts and alveolar bone loss (Marins et al., 2020), improved peri-implant bone quality and osseointegration (Li et al., 2012), and accelerated new bone formation in the expanded midpalatal suture (Kirschneck et al., 2014). Sclerostin can be used in serum or gingival biopsies in patients with chronic periodontitis (Miranda et al., 2018). Also, Sclerostin was confirmed to be found at a higher level than RANKL in the crevicular fluid of periodontitis patient peri-implant crevice fluid in patients with peri-implantitis (Yakar et al., 2019). Inhibition of sclerostin can be a new indicator of periodontitis (Balli et al., 2015). Anti-sclerostin can be used as an anti-bone resorption agent and anti-RANKL-inducing treatment by improving periodontal conditions (Koide et al., 2017). Modulation of this microenvironment may complement traditional treatments for periodontal disease and may even promote periodontal regeneration. However, excessive amounts of this inflammatory response can cause serious harm to periodontal tissue and alveolar bone. Therefore, treatment for periodontitis, an inflammatory disease, should be developed with the aim of suppressing inflammation and preventing bone loss.

Osteogenesis (bone formation through osteoblasts) and osteoclastogenesis (bone resorption through osteoclasts) are physiologically balanced during bone remodeling (Teitelbaum, 2000; Asagiri and Takayanagi, 2007). Critical cytokines such as RANKL that generate a crucial signal for osteoclast production and survival govern osteoclast development (Teitelbaum, 2000). As a result, RANKL-stimulated macrophage cells, such as BMMs or RAW264.7, have been widely used as in vitro models to assess bone resorption activity (Elisia et al., 2018). In a previous paper, we determined that RANKL-stimulated RAW264.7 could differentiate into osteoclasts, and omega-3, together with aspirin, inhibited RANKL-induced osteoclastogenesis by downregulating an osteoclast-specific gene and pro-inflammatory genes (Yang et al., 2019). Another study determined that aloe-emodin suppressed pro-inflammatory mediators such as iNOS, COX-2, and prostaglandin E2 in RAW264.7 cells in a dose-dependent manner (Park et al., 2009). LPS can activate the production of TNF-α, IL-1β, NO, and other oxidative parameters (Sheeba and Asha, 2009). Moreover, abnormal expression of TNF-α, IL-1β, and NO is associated with chronic inflammatory diseases (Soufli et al., 2016). Therefore, suppressing pro-inflammatory cytokines is essential for treating immune disorders. Even though numerous anti-bone resorption agents have been developed and are currently in use as mentioned above, none is as effective and widely used as bisphosphonates such as alendronate.

This study found that orally administered CBD and taurine combination inhibited bone loss in a pathogen-stimulated periodontitis animal. Combining CBD with taurine significantly decreased the probing pocket depths and bone resorption compared to the vehicle group in the periodontitis model. This was the first study to investigate the effects of CBD and taurine on P. gingivalis–induced periodontitis in rats. In summary, the combination of CBD with taurine significantly reduced inflammatory cytokines and bone resorption and recovered pocket depth and ABC-CEJ distance, suggesting that combining CBD with taurine could be a novel therapeutic agent against periodontal disease as well as bone lytic diseases such as osteoporosis.

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (NRF2021R1I1A3055927 to Soh Y), and by research funds of Jeonbuk National University in 2024. This paper was also supported by a grant from the Technology Innovation Program (20012892) funded by the Ministry of Trade, Industry & Energy (MOTIE, Korea).

The authors declare that there is no conflict of interest.